Pyrrolobenzodiazepines and targeted conjugates

ABSTRACT

This invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepine dimers having a C2-C3 double bond and an aryl group at the C2 position in each monomer unit, and their inclusion in targeted conjugates. The differing substituent groups may offer advantages in the preparation and use of the compounds, particularly in their biological properties and the synthesis of conjugates, and the biological properties of these conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 14/351,172 filedApr. 11, 2014, which is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/US2012/059870 filed Oct. 12, 2012, andclaims the benefit of U.S. Provisional Application No. 61/547,195 filedOct. 14, 2011, which is incorporated by reference herein.

The present invention relates to pyrrolobenzodiazepines (PBDs), inparticular pyrrolobenzodiazepine dimers having a C2-C3 double bond andan aryl group at the C2 position in each monomer unit, and theirinclusion in targeted conjugates.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, andnumerous synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. andThurston, D. E., Chem. Rev. 2011 111 (4), 2815-2864). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11 position which is the electrophilic centre responsiblefor alkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11(1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237(1986)). Their ability to form an adduct in the minor groove, enablesthem to interfere with DNA processing, hence their use as antitumouragents.

It has been previously disclosed that the biological activity of thesemolecules can be potentiated by joining two PBD units together throughtheir C8/C′-hydroxyl functionalities via a flexible alkylene linker(Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992);Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBDdimers are thought to form sequence-selective DNA lesions such as thepalindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., etal., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al.,Biochemistry, 44, 4135-4147) which is thought to be mainly responsiblefor their biological activity. One example of a PBD dimmer, SG2000(SJG-136):

has recently entered Phase II clinical trials in the oncology area(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C.,et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al.,Cancer Research, 64, 6693-6699 (2004)).

More recently, the present inventors have previously disclosed in WO2005/085251, dimeric PBD compounds bearing C2 aryl substituents, such asSG2202 (ZC-207):

and in WO2006/111759, bisulphites of such PBD compounds, for exampleSG2285 (ZC-423):

These compounds have been shown to be highly useful cytotoxic agents(Howard, P. W., et al., Bioorg. Med. Chem. (2009), 19 (22), 6463-6466,doi: 10.1016/j.bmc1.2009.09.012).

Due to the manner in which these highly potent compounds act incross-linking DNA, these molecules have been made symmetrically. Thisprovides for straightforward synthesis, either by constructing the PBDmoieties simultaneously having already formed the dimer linkage, or byreacting already constructed PBD moieties with the dimer linking group.

WO 2010/043880 discloses unsymmetrical dimeric PBD compound bearing arylgroups in the C2 position of each monomer, where one of these arylgroups bears a substituent designed to provide an anchor for linking thecompound to another moiety. Co-pending International applicationPCT/US2011/032664, filed 15 Apr. 2011, discloses the inclusion of thesePBD dimer compounds in targeted conjugates.

DISCLOSURE OF THE INVENTION

The present inventors have developed further unsymmetrical dimeric PBDcompounds for inclusion in targeted conjugates, where the substituentson the C2 aryl group not bearing the anchor for linking the compound toanother moiety are different to those previously described. Thesediffering substituent groups may offer advantages in the preparation anduse of the compounds, particularly in their biological properties andthe synthesis of conjugates, and the biological properties of theseconjugates.

The present invention comprises a compound with the formula I:

or a pharmaceutically acceptable salt or solvate thereof,wherein:R² is of formula III:

where A is a C₅₋₇ aryl group, X is

or NHR^(N), wherein R^(N) is selected from the group comprising H andC₁₋₄ alkyl and either(i) Q¹ is a single bond, and Q² is selected from a single bond and—Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH and nis from 1 to 3; or(ii) Q¹ is —CH═CH—, and Q² is a single bond;R¹² is a C₅₋₁₀ aryl group, substituted by a group selected from OH,CO₂H, CO₂R^(O), where R^(O) is selected from C₁₋₄ alkyl;R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, nitro, Me₃Sn and halo;where R and R′ are independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups;R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Snand halo; either:(a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl; or(b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogenand carbon atoms to which they are bound; or(c) R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3 and M is a monovalentpharmaceutically acceptable cation;R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄ alkyl),and/or aromatic rings, e.g. benzene or pyridine;Y and Y′ are selected from O, S, or NH;R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ andR⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹,wherein if R¹¹ and R^(11′) are SO_(z)M, M may represent a divalentpharmaceutically acceptable cation.

A second aspect of the present invention provides the use of a compoundof the first aspect of the invention in the manufacture of a medicamentfor treating a proliferative disease. The second aspect also provides acompound of the first aspect of the invention for use in the treatmentof a proliferative disease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

A third aspect of the present invention comprises a compound of formulaII:

or a pharmaceutically acceptable salt or solvate thereof,wherein:R² is of formula III:

where A is a C₅₋₇ aryl group, X is

or NHR^(N), wherein R^(N) is selected from the group comprising H andC₁₋₄ alkyl and either(i) Q¹ is a single bond, and Q² is selected from a single bond and—Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH and nis from 1 to 3; or(ii) Q¹ is —CH═CH—, and Q² is a single bond;R¹² is a C₅₋₁₀ aryl group, substituted by a group selected from OH,CO₂H, CO₂R^(O), where R^(O) is selected from C₁₋₄ alkyl;R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, nitro, Me₃Sn and halo;where R and R′ are independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups;R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Snand halo; either:(a) R¹⁰ is carbamate nitrogen protecting group, and R¹¹ is O-Prot^(O),wherein Prot^(O) is an oxygen protecting group; or(b) R¹⁰ is a hemi-aminal nitrogen protecting group and R¹¹ is an oxogroup;R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄ alkyl),and/or aromatic rings, e.g. benzene or pyridine;Y and Y′ are selected from O, S, or NH;R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ andR⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹.

A fourth aspect of the present invention comprises a method of making acompound of formula I, or a pharmaceutically acceptable salt or solvatethereof, from a compound of formula II, or a pharmaceutically acceptablesalt or solvate thereof, by deprotection of the imine bond.

The unsymmetrical dimeric PBD compounds of the present invention aremade by different strategies to those previously employed in makingsymmetrical dimeric PBD compounds. In particular, the present inventorshave developed a method which involves adding each each C2 substituentto a symmetrical PBD dimer core in separate method steps. Accordingly, afifth aspect of the present invention provides a method of making acompound of the first or third aspect of the invention, comprising atleast one of the method steps set out below.

In a sixth aspect, the present invention relates to Conjugatescomprising dimers of PBDs linked to a targeting agent, wherein the PBDdimer is of formula I, or a pharmaceutically acceptable salt or solvatethereof (supra).

In some embodiments, the Conjugates have the following formula IV:

L-(LU-D)_(p)  (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), LU is a Linker unit and D is aDrug unit that is a PBD dimer (see below).

The subscript p is from 1 to 20. Accordingly, the Conjugates comprise aLigand unit covalently linked to at least one Drug unit by a Linkerunit. The Ligand unit, described more fully below, is a targeting agentthat binds to a target moiety. The Ligand unit can, for example,specifically bind to a cell component (a Cell Binding Agent) or to othertarget molecules of interest. Accordingly, the present invention alsoprovides methods for the treatment of, for example, various cancers andautoimmune disease. These methods encompass the use of the Conjugateswherein the Ligand unit is a targeting agent that specifically binds toa target molecule. The Ligand unit can be, for example, a protein,polypeptide or peptide, such as an antibody, an antigen-binding fragmentof an antibody, or other binding agent, such as an Fc fusion protein.

In the conjugates of the present invention, the PBD dimer D is offormula I, or a pharmaceutically acceptable salt or solvate thereof,except that X is

wherein R^(N) is selected from the group comprising H and C₁₋₄ alkyl,and the asterix indicates the point of attachment to the remainder ofthe Drug unit and the wavy line indicates the point of attachment to theLinker Unit.

The drug loading is represented by p, the number of drug molecules perLigand unit (e.g., an antibody). Drug loading may range from 1 to 20Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, prepresents the average drug loading of the Conjugates in thecomposition, and p ranges from 1 to 20.

In some embodiments, p is from about 1 to about 8 Drug units per Ligandunit. In some embodiments, p is 1. In some embodiments, p is 2. In someembodiments, p is from about 2 to about 8 Drug units per Ligand unit. Insome embodiments, p is from about 2 to about 6, 2 to about 5, or 2 toabout 4 Drug units per Ligand unit. In some embodiments, p is about 2,about 4, about 6 or about 8 Drug units per Ligand unit.

The average number of Drugs units per Ligand unit in a preparation froma conjugation reaction may be characterized by conventional means suchas mass spectroscopy, ELISA assay, and HPLC. The quantitativedistribution of Conjugates in terms of p may also be determined. In someinstances, separation, purification, and characterization of homogeneousConjugates, where p is a certain value, from Conjugates with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

In a seventh aspect, the present invention relates to Linker-Drugcompounds (i.e., Drug-Linkers) comprising dimers of PBDs (see above)linked to a linking unit. These Drug-linkers can be used asintermediates for the synthesis of Conjugates comprising dimers of PBDslinked to a targeting agent.

These Drug-Linkers have the following formula V:

LU-D  (V)

or a pharmaceutically acceptable salt or solvate thereof, wherein LU isa Linker unit and D is a Drug unit that is a PBD dimer.

In the Drug-Linkers of the present invention, the PBD dimer D is offormula I, or a pharmaceutically acceptable salt or solvate thereof,except that X is is

wherein R^(N) is selected from the group comprising H and C₁₋₄ alkyl,and the asterix indicates the point of attachment to the remainder ofthe Drug unit and the wavy line indicates the point of attachment to theLinker Unit.

FIGURES

FIG. 1 shows the effect on tumour volume of a conjugate of the presentinvention at two different doses;

FIG. 2 shows the effect on tumour volume of the same conjugate as inFIG. 1 on a different tumour.

DEFINITIONS Pharmaceutically Acceptable Cations

Examples of pharmaceutically acceptable monovalent and divalent cationsare discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), whichis incorporated herein by reference in its entirety and for allpurposes.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cationsinclude, but are not limited to, alkali metal ions such as Na⁺ and K⁺.Examples of pharmaceutically acceptable divalent inorganic cationsinclude, but are not limited to, alkaline earth cations such as Ca²⁺andMg²⁺. Examples of pharmaceutically acceptable organic cations include,but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substitutedammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). The term “C₁₋₄ alkyl”as used herein, pertains to a monovalent moiety obtained by removing ahydrogen atom from a carbon atom of a hydrocarbon compound having from 1to 4 carbon atoms, which may be aliphatic or alicyclic, and which may besaturated or unsaturated (e.g. partially unsaturated, fullyunsaturated). Similarly, the term “C₁₋₂alkyl” as used herein, pertainsto a monovalent moiety obtained by removing a hydrogen atom from acarbon atom of a hydrocarbon compound having from 1 to 2 carbon atoms,i.e. methyl or ethyl.

Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl,cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:        cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),        cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),        dimethylcyclopropane (C₅), methylcyclobutane (C₅),        dimethylcyclobutane (C₆), methylcyclopentane (C₆),        dimethylcyclopentane (C₇) and methylcyclohexane (C₇);    -   unsaturated monocyclic hydrocarbon compounds:        cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),        cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene        (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),        methylcyclopentene (C₆), dimethylcyclopentene (C₇) and        methylcyclohexene (C₇); and    -   saturated polycyclic hydrocarbon compounds:        norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);O₃: trioxane (C₆);N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);N₂O₁: oxadiazine (C₆);O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. The term “C₅₋₇ aryl”, as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from an aromatic ring atomof an aromatic compound, which moiety has from 5 to 7 ring atoms and theterm “C₅₋₁₀ aryl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 5 to 10 ring atoms. Preferably,each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, C₅₋₁₀, etc.)denote the number of ring atoms, or range of number of ring atoms,whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl”as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);O₁: furan (oxole) (C₅);S₁: thiophene (thiole) (C₅);N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);N₂O₁: oxadiazole (furazan) (C₅);N₃O₁: oxatriazole (C₅);N₁S₁: thiazole (C₅), isothiazole (C₅);N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);N₃: triazole (C₅), triazine (C₆); and,N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene        (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁),        phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),        thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),        phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groupsinclude, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇alkylacyl or C₁₋₇alkanoyl), aC₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carbon/late, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyl,and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and ═NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples of C₁₋₇alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate). Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxysubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinyloxy groups include, but are not limited to, —OS(═O)CH₃ and—OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groupsinclude, but are not limited to, —OS(═O)₂CH₃ (mesylate) and—OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂,—OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to abidentate moiety obtained by removing two hydrogen atoms, either bothfrom the same carbon atom, or one from each of two different carbonatoms, of a hydrocarbon compound having from 3 to 12 carbon atoms(unless otherwise specified), which may be aliphatic or alicyclic, andwhich may be saturated, partially unsaturated, or fully unsaturated.Thus, the term “alkylene” includes the sub-classes alkenylene,alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are notlimited to, —(CH₂)_(n)— where n is an integer from 3 to 12, for example,—CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂—(pentylene) and —CH₂CH₂CH₂CH—₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂ alkylene groups include, but arenot limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—,—CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and—CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene, and alkynylene groups) include, but are not limited to,—CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—,—CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—,—CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—C≡C—CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene and alkynylene groups) include, but are not limited to,—C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C—CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentylene (e.g.cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene;3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Oxygen protecting group: the term “oxygen protecting group” refers to amoiety which masks a hydroxy group, and these are well known in the art.A large number of suitable groups are described on pages 23 to 200 ofGreene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis,3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporatedherein by reference in its entirety and for all purposes. Classes ofparticular interest include silyl ethers (e.g. TMS, TBDMS), substitutedmethyl ethers (e.g. THP) and esters (e.g. acetate).

Carbamate nitrogen protecting group: the term “carbamate nitrogenprotecting group” pertains to a moiety which masks the nitrogen in theimine bond, and these are well known in the art. These groups have thefollowing structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference in itsentirety and for all purposes.

Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogenprotecting group” pertains to a group having the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference in its entirety and for all purposes.

Conjugates

The present invention provides Conjugates comprising a PBD dimerconnected to a Ligand unit via a Linker unit. In one embodiment, theLinker unit includes a Stretcher unit (A), a Specificity unit (L¹), anda Spacer unit (L²). The Linker unit is connected at one end to theLigand unit (L) and at the other end to the PBD dimer compound (D).

In one aspect, such a Conjugate is shown below in formula IVa:

L-(A¹ _(a)-L¹ _(s)-L² _(y)-D)_(p)  (IVa)

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)-L² _(y)- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit,    -   a is 1 or 2,    -   -L¹- is a Specificity unit,    -   s is an integer ranging from 0 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is a PBD dimer; and    -   p is from 1 to 20.

In another aspect, such a Conjugate is shown below in formula IVb:

Also illustrated as:

L-(A¹ _(a)-L² _(y)(-L¹ _(s))-D)_(p)  (IVb)

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)(L² _(y))- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit linked to a Stretcher unit (L²),    -   a is 1 or 2,    -   -L¹- is a Specificity unit linked to a Stretcher unit (L²),    -   s is an integer ranging from 0 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is a PBD dimer; and    -   p is from 1 to 20.

Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In one embodiment, the Conjugate has the formula:

L-(A¹ _(a)-L¹ _(s)L² _(y)-D)_(p)

L-(A¹ _(a)-L¹ _(s)D)_(p)

L-(A¹-L¹-D)_(p) or

L-(A¹-D)_(p)

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein L, A¹, a L¹, s, L², D, y and p are as described above.

In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit, which is a covalent bond, a        self-immolative group or together with —OC(═O)— forms a        self-immolative group, and L² is optional. —OC(═O)— may be        considered as being part of L¹ or L², as appropriate.

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

CBA-A¹ _(a)-L¹ _(s)-L² _(y)-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit which is a covalent bond or a        self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is        0 or 1 or 2.

In the embodiments illustrated above, L¹ can be a cleavable Specificityunit, and may be referred to as a “trigger” that when cleaved activatesa self-immolative group (or self-immolative groups) L², when aself-immolative group(s) is present. When the Specificity unit L¹ iscleaved, or the linkage (i.e., the covalent bond) between L¹ and L² iscleaved, the self-immolative group releases the Drug unit (D).

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        (D), CBA is the Cell Binding Agent, L¹ is a Specificity unit        connected to L², A¹ is a Stretcher unit connecting L² to the        Cell Binding Agent, L² is a self-immolative group, and a is 1 or        2, s is 1 or 2, and y is 1 or 2.

In the various embodiments discussed herein, the nature of L¹ and L² canvary widely. These groups are chosen on the basis of theircharacteristics, which may be dictated in part, by the conditions at thesite to which the conjugate is delivered. Where the Specificity unit L¹is cleavable, the structure and/or sequence of L¹ is selected such thatit is cleaved by the action of enzymes present at the target site (e.g.,the target cell). L¹ units that are cleavable by changes in pH (e.g.acid or base labile), temperature or upon irradiation (e.g. photolabile)may also be used. L¹ units that are cleavable under reducing oroxidising conditions may also find use in the Conjugates.

In some embodiments, L¹ may comprise one amino acid or a contiguoussequence of amino acids. The amino acid sequence may be the targetsubstrate for an enzyme.

In one embodiment, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase. For example, L¹may be cleaved by a lysosomal protease, such as a cathepsin.

In one embodiment, L² is present and together with —C(═O)O— forms aself-immolative group or self-immolative groups. In some embodiments,—C(═O)O— also is a self-immolative group.

In one embodiment, where L¹ is cleavable by the action of an enzyme andL² is present, the enzyme cleaves the bond between L¹ and L², wherebythe self-immolative group(s) release the Drug unit.

L¹ and L², where present, may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH, and    -   —O— (a glycosidic bond).

An amino group of L¹ that connects to L² may be the N-terminus of anamino acid or may be derived from an amino group of an amino acid sidechain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to L² may be the C-terminus of anamino acid or may be derived from a carboxyl group of an amino acid sidechain, for example a glutamic acid amino acid side chain.

A hydroxy group of L¹ that connects to L² may be derived from a hydroxygroup of an amino acid side chain, for example a serine amino acid sidechain.

In one embodiment, —C(═O)O— and L² together form the group:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to the L¹,        Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The        phenylene ring is optionally substituted with one, two or three        substituents as described herein.

In one embodiment, Y is NH.

In one embodiment, n is 0 or 1. Preferably, n is 0.

Where Y is NH and n is 0, the self-immolative group may be referred toas a p-aminobenzylcarbonyl linker (PABC).

The self-immolative group will allow for release of the Drug unit (i.e.,the asymmetric PBD) when a remote site in the linker is activated,proceeding along the lines shown below (for n=0):

-   -   where the asterisk indicates the attachment to the Drug, L* is        the activated form of the remaining portion of the linker and        the released Drug unit is not shown. These groups have the        advantage of separating the site of activation from the Drug.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above. Each phenylene ring is optionally substituted with one,        two or three substituents as described herein. In one        embodiment, the phenylene ring having the Y substituent is        optionally substituted and the phenylene ring not having the Y        substituent is unsubstituted.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or        CR.

In one embodiment, D is N.

In one embodiment, D is CH.

In one embodiment, E is O or S.

In one embodiment, F is CH.

In a preferred embodiment, the covalent bond between L¹ and L² is acathepsin labile (e.g., cleavable) bond.

In one embodiment, L¹ comprises a dipeptide. The amino acids in thedipeptide may be any combination of natural amino acids and non-naturalamino acids. In some embodiments, the dipeptide comprises natural aminoacids. Where the linker is a cathepsin labile linker, the dipeptide isthe site of action for cathepsin-mediated cleavage. The dipeptide thenis a recognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys-, Val-Cit or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those described byDubowchik et al., which is incorporated herein by reference in itsentirety and for all purposes.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below. Protected amino acid sequences are cleavable byenzymes. For example, a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those aminoacids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;    -   Asn: Trt, Xan;    -   Asp: Bzl, t-Bu;    -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;    -   Glu: Bzl, t-Bu;    -   Gln: Trt, Xan;    -   His: Boc, Dnp, Tos, Trt;    -   Lys: Boc, Z—Cl, Fmoc, Z;    -   Ser: Bzl, TBDMS, TBDPS;    -   Thr: Bz;    -   Trp: Boc;    -   Tyr: Bzl, Z, Z—Br.

In one embodiment, —X₂— is connected indirectly to the Drug unit. Insuch an embodiment, the Spacer unit L² is present.

In one embodiment, —X₂— is connected directly to the Drug unit. In suchan embodiment, the Spacer unit L² is absent.

In one embodiment, the dipeptide is used in combination with aself-immolative group(s) (the Spacer unit). The self-immolative group(s)may be connected to —X₂—.

Where a self-immolative group is present, —X₂— is connected directly tothe self-immolative group. In one embodiment, —X₂— is connected to thegroup Y of the self-immolative group. Preferably the group —X₂—CO— isconnected to Y, where Y is NH.

In one embodiment, —X₁— is connected directly to A¹. Preferably thegroup NH—X₁— (the amino terminus of X₁) is connected to A¹. A¹ maycomprise the functionality —CO— thereby to form an amide link with —X₁—.

In one embodiment, L¹ and L² together with —OC(═O)— comprise the group—X₁—X₂-PABC-. The PABC group is connected directly to the Drug unit. Inone example, the self-immolative group and the dipeptide together formthe group -Phe-Lys-PABC-, which is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of L¹ or the point of attachment to A¹.        Preferably, the wavy line indicates the point of attachment to        A¹.

Alternatively, the self-immolative group and the dipeptide together formthe group -Val-Ala-PABC-, which is illustrated below:

-   -   where the asterisk and the wavy line are as defined above.

In another embodiment, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A¹, Y        is a covalent bond or a functional group, and E is a group that        is susceptible to cleavage thereby to activate a self-immolative        group.

E is selected such that the group is susceptible to cleavage, e.g., bylight or by the action of an enzyme. E may be —NO₂ or glucuronic acid(e.g., β-glucuronic acid). The former may be susceptible to the actionof a nitroreductase, the latter to the action of a β-glucuronidase.

The group Y may be a covalent bond.

The group Y may be a functional group selected from:

-   -   —C(═O)—    -   —NH—    -   —O—    -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   SO₂, and    -   —S—.

The group Y is preferably —NH—, —CH₂—, —O—, and —S—.

In some embodiments, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A, Y is        a covalent bond or a functional group and E is glucuronic acid        (e.g., β-glucuronic acid). Y is preferably a functional group        selected from —NH—.

In some embodiments, L¹ and L² together represent:

-   -   where the asterisk indicates the point of attachment to the        remainder of L² or the Drug unit, the wavy line indicates the        point of attachment to A¹, Y is a covalent bond or a functional        group and E is glucuronic acid (e.g., β-glucuronic acid). Y is        preferably a functional group selected from —NH—, —CH₂—, —O—,        and —S—.

In some further embodiments, Y is a functional group as set forth above,the functional group is linked to an amino acid, and the amino acid islinked to the Stretcher unit A¹. In some embodiments, amino acid isβ-alanine. In such an embodiment, the amino acid is equivalentlyconsidered part of the Stretcher unit.

The Specificity unit L¹ and the Ligand unit are indirectly connected viathe Stretcher unit.

L¹ and A¹ may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the connection between the Ligand unit and A¹ isthrough a thiol residue of the Ligand unit and a maleimide group of A¹.

In one embodiment, the connection between the Ligand unit and A¹ is:

-   -   where the asterisk indicates the point of attachment to the        remaining portion of A¹, L¹, L² or D, and the wavy line        indicates the point of attachment to the remaining portion of        the Ligand unit. In this embodiment, the S atom is typically        derived from the Ligand unit.

In each of the embodiments above, an alternative functionality may beused in place of the malemide-derived group shown below:

-   -   where the wavy line indicates the point of attachment to the        Ligand unit as before, and the asterisk indicates the bond to        the remaining portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the wavy line indicates point of attachment to the Ligand        unit, and the asterisk indicates the bond to the remaining        portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with a Ligand unit (e.g., a Cell BindingAgent), is selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   —S—,    -   —S—S—,    -   —CH₂C(═O)—    -   —C(═O)CH₂—,    -   ═N—NH—, and    -   —NH—N═.

Of these —C(═O)CH₂— may be preferred especially when the carbonyl groupis bound to —NH—.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the Ligand unit, is selected from:

-   -   where the wavy line indicates either the point of attachment to        the Ligand unit or the bond to the remaining portion of the A¹        group, and the asterisk indicates the other of the point of        attachment to the Ligand unit or the bond to the remaining        portion of the A¹ group.

Other groups suitable for connecting L¹ to the Cell Binding Agent aredescribed in WO 2005/082023.

In one embodiment, the Stretcher unit A¹ is present, the Specificityunit L¹ is present and Spacer unit L² is absent. Thus, L¹ and the Drugunit are directly connected via a bond. Equivalently in this embodiment,L² is a bond.

L¹ and D may be connected by a bond selected from:

-   -   —C(═O)N<,    -   —OC(═O)N<, and    -   —NHC(═O)N<,        where N< is part of D.

In one embodiment, L¹ and D are preferably connected by a bond:

-   -   —C(═O)N<.

In one embodiment, L¹ comprises a dipeptide and one end of the dipeptideis linked to D. As described above, the amino acids in the dipeptide maybe any combination of natural amino acids and non-natural amino acids.In some embodiments, the dipeptide comprises natural amino acids. Wherethe linker is a cathepsin labile linker, the dipeptide is the site ofaction for cathepsin-mediated cleavage. The dipeptide then is arecognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys- or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those describedabove.

In one embodiment, L¹-D is:

-   -   where —NH—X₁—X₂—CO is the dipeptide, —N< is part of the Drug        unit, the asterisk indicates the points of attachment to the        remainder of the Drug unit, and the wavy line indicates the        point of attachment to the remaining portion of L¹ or the point        of attachment to A¹. Preferably, the wavy line indicates the        point of attachment to A¹.

In one embodiment, the dipeptide is valine-alanine and L¹-D is:

-   -   where the asterisks, —N< and the wavy line are as defined above.

In one embodiment, the dipeptide is phenylalanine-lysine and L¹-D is:

-   -   where the asterisks, —N< and the wavy line are as defined above.

In one embodiment, the dipeptide is valine-citrulline.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most        preferably 3 or 7.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A¹-L¹ is:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the rest of the Ligand unit, and n is        0 to 6. In one embodiment, n is 5.

In one embodiment, the group L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the remainder of the Ligand unit, and        n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the remainder of the Ligand unit, n        is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the Stretcher unit is an acetamide unit, having theformula:

-   -   where the asterisk indicates the point of attachment to the        remainder of the Stretcher unit, L¹ or D, and the wavy line        indicates the point of attachment to the Ligand unit.

Linker-Drugs

In other embodiments, Linker-Drug compounds are provided for conjugationto a Ligand unit. In one embodiment, the Linker-Drug compounds aredesigned for connection to a Cell Binding Agent.

In one embodiment, the Drug Linker compound has the formula:

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D, as defined above), G¹ is a Stretcher group (A¹) to form        a connection to a Ligand unit, L¹ is a Specificity unit, L² (a        Spacer unit) is a covalent bond or together with —OC(═O)— forms        a self-immolative group(s).

In another embodiment, the Drug Linker compound has the formula:

G¹-L¹-L²-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), G¹ is a Stretcher unit (A¹) to form a connection to a        Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a        covalent bond or a self-immolative group(s).

L¹ and L² are as defined above. References to connection to A¹ can beconstrued here as referring to a connection to G¹.

In one embodiment, where L¹ comprises an amino acid, the side chain ofthat amino acid may be protected. Any suitable protecting group may beused. In one embodiment, the side chain protecting groups are removablewith other protecting groups in the compound, where present. In otherembodiments, the protecting groups may be orthogonal to other protectinggroups in the molecule, where present.

Suitable protecting groups for amino acid side chains include thosegroups described in the Novabiochem Catalog 2006/2007. Protecting groupsfor use in a cathepsin labile linker are also discussed in Dubowchik etal.

In certain embodiments of the invention, the group L¹ includes a Lysamino acid residue. The side chain of this amino acid may be protectedwith a Boc or Alloc protected group. A Boc protecting group is mostpreferred.

The functional group G¹ forms a connecting group upon reaction with aLigand unit (e.g., a cell binding agent.

In one embodiment, the functional group G¹ is or comprises an amino,carboxylic acid, hydroxy, thiol, or maleimide group for reaction with anappropriate group on the Ligand unit. In a preferred embodiment, G¹comprises a maleimide group.

In one embodiment, the group G¹ is an alkyl maleimide group. This groupis suitable for reaction with thiol groups, particularly cysteine thiolgroups, present in the cell binding agent, for example present in anantibody.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most        preferably 4 or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most        preferably 4 or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most        preferably 4 or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most        preferably 4 or 8.

In each of the embodiments above, an alternative functionality may beused in place of the malemide group shown below:

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide group is replaced with a group selectedfrom:

-   -   —C(═O)OH,    -   —OH,    -   —NH₂,    -   —SH,    -   —C(═O)CH₂X, where X is Cl, Br or I,    -   —CHO,    -   —NHNH₂    -   —C≡CH, and    -   —N₃ (azide).

Of these, —C(═O)CH₂X may be preferred, especially when the carbonylgroup is bound to —NH—.

In one embodiment, L¹ is present, and G¹ is —NH₂, —NHMe, —COOH, —OH or—SH.

In one embodiment, where L¹ is present, G¹ is —NH₂ or —NHMe. Eithergroup may be the N-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is —NH₂, and L¹ is an amino acidsequence —X₁—X₂—, as defined above.

In one embodiment, L¹ is present and G¹ is COOH. This group may be theC-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is OH.

In one embodiment, L¹ is present and G¹ is SH.

The group G¹ may be convertable from one functional group to another. Inone embodiment, L¹ is present and G¹ is —NH₂. This group is convertableto another group G¹ comprising a maleimide group. For example, the group—NH₂ may be reacted with an acids or an activated acid (e.g.,N-succinimide forms) of those G¹ groups comprising maleimide shownabove.

The group G¹ may therefore be converted to a functional group that ismore appropriate for reaction with a Ligand unit.

As noted above, in one embodiment, L¹ is present and G¹ is —NH₂, —NHMe,—COOH, —OH or —SH. In a further embodiment, these groups are provided ina chemically protected form. The chemically protected form is thereforea precursor to the linker that is provided with a functional group.

In one embodiment, G¹ is —NH₂ in a chemically protected form. The groupmay be protected with a carbamate protecting group. The carbamateprotecting group may be selected from the group consisting of:

-   -   Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.

Preferably, where G¹ is —NH₂, it is protected with an Alloc or Fmocgroup.

In one embodiment, where G¹ is —NH₂, it is protected with an Fmoc group.

In one embodiment, the protecting group is the same as the carbamateprotecting group of the capping group.

In one embodiment, the protecting group is not the same as the carbamateprotecting group of the capping group. In this embodiment, it ispreferred that the protecting group is removable under conditions thatdo not remove the carbamate protecting group of the capping group.

The chemical protecting group may be removed to provide a functionalgroup to form a connection to a Ligand unit. Optionally, this functionalgroup may then be converted to another functional group as describedabove.

In one embodiment, the active group is an amine. This amine ispreferably the N-terminal amine of a peptide, and may be the N-terminalamine of the preferred dipeptides of the invention.

The active group may be reacted to yield the functional group that isintended to form a connection to a Ligand unit.

In other embodiments, the Linker unit is a precursor to the Linker uithaving an active group. In this embodiment, the Linker unit comprisesthe active group, which is protected by way of a protecting group. Theprotecting group may be removed to provide the Linker unit having anactive group.

Where the active group is an amine, the protecting group may be an amineprotecting group, such as those described in Green and Wuts.

The protecting group is preferably orthogonal to other protectinggroups, where present, in the Linker unit.

In one embodiment, the protecting group is orthogonal to the cappinggroup. Thus, the active group protecting group is removable whilstretaining the capping group. In other embodiments, the protecting groupand the capping group is removable under the same conditions as thoseused to remove the capping group.

In one embodiment, the Linker unit is:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of the Linker unit, as applicable or the point        of attachment to G¹. Preferably, the wavy line indicates the        point of attachment to G¹.

In one embodiment, the Linker unit is:

where the asterisk and the wavy line are as defined above.

Other functional groups suitable for use in forming a connection betweenL¹ and the Cell Binding Agent are described in WO 2005/082023.

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide,peptide and a non-peptidic agent that specifically binds to a targetmolecule. In some embodiments, the Ligand unit may be a protein,polypeptide or peptide. In some embodiments, the Ligand unit may be acyclic polypeptide. These Ligand units can include antibodies or afragment of an antibody that contains at least one targetmolecule-binding site, lymphokines, hormones, growth factors, or anyother cell binding molecule or substance that can specifically bind to atarget. The ligand Unit is also referred to herein as a “binding agent”or “targeting agent”.

The terms “specifically binds” and “specific binding” refer to thebinding of an antibody or other protein, polypeptide or peptide to apredetermined molecule (e.g., an antigen).

Typically, the antibody or other molecule binds with an affinity of atleast about 1×10⁷ M⁻¹, and binds to the predetermined molecule with anaffinity that is at least two-fold greater than its affinity for bindingto a non-specific molecule (e.g., BSA, casein) other than thepredetermined molecule or a closely-related molecule.

Examples of Ligand units include those agents described for use in WO2007/085930, which is incorporated by reference herein in its entiretyand for all purposes.

In some embodiments, the Ligand unit is a Cell Binding Agent that bindsto an extracellular target on a cell. Such a Cell Binding Agent can be aprotein, polypeptide, peptide or a non-peptidic agent. In someembodiments, the Cell Binding Agent may be a protein, polypeptide orpeptide. In some embodiments, the Cell Binding Agent may be a cyclicpolypeptide. The Cell Binding Agent also may be antibody or anantigen-binding fragment of an antibody. Thus, in one embodiment, thepresent invention provides an antibody-drug conjugate (ADC).

In one embodiment the antibody is a monoclonal antibody; chimericantibody; humanized antibody; fully human antibody; or a single chainantibody. One embodiment the antibody is a fragment of one of theseantibodies having biological activity. Examples of such fragmentsinclude Fab, Fab′, F(ab′)₂ and Fv fragments.

The antibody may be a diabody, a domain antibody (DAB) or a single chainantibody.

In one embodiment, the antibody is a monoclonal antibody.

Antibodies for use in the present invention include those antibodiesdescribed in WO 2005/082023 which is incorporated by reference herein inits entirety and for all purposes. Particularly preferred are thoseantibodies for tumour-associated antigens. Examples of those antigensknown in the art include, but are not limited to, thosetumour-associated antigens set out in WO 2005/082023. See, for instance,pages 41-55.

In some embodiments, the conjugates are designed to target tumour cellsvia their cell surface antigens. The antigens may be cell surfaceantigens which are either over-expressed or expressed at abnormal timesor cell types. Preferably, the target antigen is expressed only onproliferative cells (preferably tumour cells); however this is rarelyobserved in practice. As a result, target antigens are usually selectedon the basis of differential expression between proliferative andhealthy tissue.

Antibodies have been raised to target specific tumour related antigensincluding:

-   -   Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg,        Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA        (prostate specific membrane antigen), BCMA, E-selectin, EphB2,        Melanotransferin, Muc16 and TMEFF2. In any of the embodiments        provided herein, the Ligand unit can be a monoclonal antibody        that specifically binds to the Cripto antigen, CD19 antigen,        CD20 antigen, CD22 antigen, CD30 antigen, CD33 antigen,        Glycoprotein NMB, CanAg antigen, Her2 (ErbB2/Neu) antigen, CD56        (NCAM) antigen, CD70 antigen, CD79 antigen, CD138 antigen, PSCA,        PSMA (prostate specific membrane antigen), BCMA, E-selectin,        EphB2, Melanotransferin, Muc16 antigen or TMEFF2 antigen.

The Ligand unit is connected to the Linker unit. In one embodiment, theLigand unit is connected to A, where present, of the Linker unit.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a thioether bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a disulfide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an amide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an ester bond.

In one embodiment, the connection between the Ligand unit and the Linkeris formed between a thiol group of a cysteine residue of the Ligand unitand a maleimide group of the Linker unit.

The cysteine residues of the Ligand unit may be available for reactionwith the functional group of the Linker unit to form a connection. Inother embodiments, for example where the Ligand unit is an antibody, thethiol groups of the antibody may participate in interchain disulfidebonds. These interchain bonds may be converted to free thiol groups bye.g. treatment of the antibody with DTT prior to reaction with thefunctional group of the Linker unit.

In some embodiments, the cysteine residue is introduced into the heavyor light chain of an antibody. Positions for cysteine insertion bysubstitution in antibody heavy or light chains include those describedin Published U.S. Application No. 2007-0092940 and International PatentPublication WO2008/070593, which are incorporated by reference herein intheir entirety and for all purposes.

Methods of Treatment

The compounds or conjugates of the present invention may be used in amethod of therapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a compound of formula I or conjugatethereof. The term “therapeutically effective amount” is an amountsufficient to show benefit to a patient. Such benefit may be at leastamelioration of at least one symptom. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of what is being treated. Prescription of treatment, e.g.decisions on dosage, is within the responsibility of generalpractitioners and other medical doctors.

A compound or conjugate may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g. drugs; surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a compound of formula I, or conjugatethereof, a pharmaceutically acceptable excipient, carrier, buffer,stabiliser or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material will depend on the route of administration, which may beoral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The Compounds and Conjugates can be used to treat proliferative diseaseand autoimmune disease. The term “proliferative disease” pertains to anunwanted or uncontrolled cellular proliferation of excessive or abnormalcells which is undesired, such as, neoplastic or hyperplastic growth,whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Other cancers of interest include, but are not limitedto, haematological; malignancies such as leukemias and lymphomas, suchas non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone,mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers ofB or T cell origin.

Examples of autoimmune disease include the following: rheumatoidarthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis,allergic encephalomyelitis), psoriatic arthritis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Graves' disease, glomerulonephritis, autoimmune hepatologicaldisorder, inflammatory bowel disease (e.g., Crohn's disease),anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetesmellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of Blymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g.,rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments,the autoimmune disorder is a T cell-mediated immunological disorder.

In some embodiments, the amount of the Conjugate administered rangesfrom about 0.01 to about 10 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.01 to about 5mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.05 to about 5 mg/kg per dose. In someembodiments, the amount of the Conjugate administered ranges from about0.1 to about 5 mg/kg per dose. In some embodiments, the amount of theConjugate administered ranges from about 0.1 to about 4 mg/kg per dose.In some embodiments, the amount of the Conjugate administered rangesfrom about 0.05 to about 3 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.1 to about 3mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.1 to about 2 mg/kg per dose.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carbon/late) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Carbinolamines

The invention includes compounds where a solvent adds across the iminebond of the PBD moiety, which is illustrated below where the solvent iswater or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD.

The balance of these equilibria depend on the conditions in which thecompounds are found, as well as the nature of the moiety itself.

These particular compounds may be isolated in solid form, for example,by lyophilisation.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

General Synthetic Routes

The synthesis of PBD compounds is extensively discussed in the followingreferences, which discussions are incorporated herein by reference intheir entirety and for all purposes:

a) WO 00/12508 (pages 14 to 30);b) WO 2005/023814 (pages 3 to 10);c) WO 2004/043963 (pages 28 to 29); andd) WO 2005/085251 (pages 30 to 39).

Synthesis Route

The compounds of the present invention, where R¹⁰ and R¹¹ form anitrogen-carbon double bond between the nitrogen and carbon atoms towhich they are bound, can be synthesised from a compound of Formula 2:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), R¹², X, X′ and R″ are asdefined for compounds of formula I, Prot^(N) is a nitrogen protectinggroup for synthesis and Prot^(O) is a protected oxygen group forsynthesis or an oxo group, by deprotecting the imine bond by standardmethods.

The compound produced may be in its carbinolamine or carbinolamine etherform depending on the solvents used. For example if Prot^(N) is Troc andProt^(O) is an oxygen protecting group for synthesis, then thedeprotection is carried out using a Cd/Pb couple to yield the compoundof formula (I). If Prot^(N) is SEM, or an analogous group, and Prot^(O)is an an oxo group, then the oxo group can be removed by reduction,which leads to a protected carbinolamine intermediate, which can then betreated to remove the SEM protecting group, followed by the eliminationof water. The reduction of the compound of Formula 2 can be accomplishedby, for example, superhydride or lithium tetraborohydride, whilst asuitable means for removing the SEM protecting group is treatment withsilica gel.

Compounds of formula 2 can be synthesised from a compound of formula 3a:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of formula 2, by coupling an organometallicderivative comprising R¹², such as an organoboron derivative. Theorganoboron derivative may be a boronate or boronic acid.

Compounds of formula 2 can be synthesised from a compound of formula 3b:

where R¹², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of formula 2, by coupling an organometallicderivative comprising R², such as an organoboron derivative. Theorganoboron derivative may be a boronate or boronic acid.

Compounds of formulae 3a and 3b can be synthesised from a compound offormula 4:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of formula 2, by coupling about a singleequivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallicderivative, such as an organoboron derivative, comprising R² or R¹².

The couplings described above are usually carried out in the presence ofa palladium catalyst, for example Pd(PPh₃)₄, Pd(OCOCH₃)₂, PdCl₂,Pd₂(dba)₃. The coupling may be carried out under standard conditions, ormay also be carried out under microwave conditions.

The two coupling steps are usually carried out sequentially. They may becarried out with or without purification between the two steps. If nopurification is carried out, then the two steps may be carried out inthe same reaction vessel. Purification is usually required after thesecond coupling step. Purification of the compound from the undesiredby-products may be carried out by column chromatography or ion-exchangeseparation.

The synthesis of compounds of formula 4 where Prot^(O) is an oxo groupand Prot^(N) is SEM are described in detail in WO 00/12508, which isincorporated herein by reference in its entirety and for all purposes.In particular, reference is made to scheme 7 on page 24, where the abovecompound is designated as intermediate P. This method of synthesis isalso described in WO 2004/043963, which is incorporated herein byreference in its entirety and for all purposes. Further reference isalso made to the synthesis of compounds 8a and 8b in WO 2010/043880(pages 36 to 45), which is incorporated herein by reference in itsentirety and for all purposes.

The synthesis of compounds of formula 4 where Prot^(O) is a protectedoxygen group for synthesis are described in WO 2005/085251, whichsynthesis is herein incorporated by reference.

Compounds of formula I where R¹⁰ and R^(10′) are H and R¹¹ and R^(11′)are SO_(z)M, can be synthesised from compounds of formula I where R¹⁰and R¹¹ form a nitrogen-carbon double bond between the nitrogen andcarbon atoms to which they are bound, by the addition of the appropriatebisulphite salt or sulphinate salt, followed by an appropriatepurification step. Further methods are described in GB 2 053 894, whichis herein incorporated by reference.

In some embodiments of the invention, particularly where R¹² bears asubstituent that is OH or CO₂H, it may be desired in the above methodsto add an organometallic derivative of R¹² where the substituent groupis protected. For example, if R¹² bears CO₂H, it may be preferred tojoin a compound where the carboxy is protected as an ester (e.g. C₁₋₄alkyl ester) and then deprotect the carboxy group at a later stage inthe synthesis. It may even be deprotected once part of the linker groupfor making a drug linker has been added. The OH substituent may beprotected by phenol protecting groups as known in the art.

Nitrogen Protecting Groups for Synthesis

Nitrogen protecting groups for synthesis are well known in the art. Inthe present invention, the protecting groups of particular interest arecarbamate nitrogen protecting groups and hemi-aminal nitrogen protectinggroups.

Carbamate nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference.

Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC,Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.

Other possible groups are nitrobenzyloxycarbonyl (e.g.4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.

Those protecting groups which can be removed with palladium catalysisare not preferred, e.g. Alloc.

Hemi-aminal nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference. The groups disclosed herein can be applied to compounds ofthe present invention. Such groups include, but are not limited to, SEM,MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, Cl₃CCH₂OCH₂—.

Protected Oxygen Group for Synthesis

Protected oxygen group for synthesis are well known in the art. A largenumber of suitable oxygen protecting groups are described on pages 23 to200 of Greene, T. W. and Wuts, G. M., Protective Groups in OrganicSynthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which isincorporated herein by reference in its entirety and for all purposes.

Classes of particular interest include silyl ethers, methyl ethers,alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates,and sulfonates.

Preferred oxygen protecting groups include acetates, TBS and THP.

Synthesis of Drug Conjugates

Conjugates comprising PBD dimers as described herein can be preparedusing the knowledge of the skilled artisan in combination with theteachings provided herein. For example, linkers are described in U.S.Pat. No. 6,214,345, U.S. Pat. No. 7,498,298 as well as WO 2009/0117531,each of which is incorporated herein by reference in their entirety andfor all purposes. Other linkers can be prepared according to thereferences cited herein or as known to the skilled artisan.

Linker-Drug compounds can be prepared according to methods known in theart in combination with the teachings provided herein. For example,linkage of amine-based X substituents (of the PBD dimer Drug unit) toactive groups of the Linker units can be performed according to methodsgenerally described in U.S. Pat. Nos. 6,214,345 and 7,498,298; and WO2009-0117531, or as otherwise known to the skilled artisan. Someexamples are shown below.

Antibodies can be conjugated to Linker-Drug compounds as described inDoronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly,antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37°C. The progress of the reaction, which reduces interchain disulfides, ismonitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) andallowed to proceed until the desired level of thiols/mAb is achieved.The reduced antibody is then cooled to 0° C. and alkylated with 1.5equivalents of maleimide drug-linker per antibody thiol. After 1 hour,the reaction is quenched by the addition of 5 equivalents of N-acetylcysteine. Quenched drug-linker is removed by gel filtration over a PD-10column. The ADC is then sterile-filtered through a 0.22 μm syringefilter. Protein concentration can be determined by spectral analysis at280 nm and 329 nm, respectively, with correction for the contribution ofdrug absorbance at 280 nm. Size exclusion chromatography can be used todetermine the extent of antibody aggregation, and RP-HPLC can be used todetermine the levels of remaining NAC-quenched drug-linker.

Antibodies with introduced cysteine residues can be conjugated toLinker-Drug compounds as described in International Patent PublicationWO2008/070593, which is incorporated by reference herein in its entiretyand for all purposes or as follows. Antibodies containing an introducedcysteine residue in the heavy chain are fully reduced by adding 10equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1MTris buffer (pH 9.0).

Following a 1 hour incubation at 37° C., the reaction is cooled to 22°C. and 30 equivalents of dehydroascorbic acid is added to selectivelyreoxidize the native disulfides, while leaving the introduced cysteinein the reduced state. The pH is adjusted to 6.5 with 1M Tris buffer (pH3.7) and the reaction is allowed to proceed for 1 hour at 22° C. The pHof the solution is then raised again to 7.4 by addition of 1 M Trisbuffer (pH 9.0). 3.5 equivalents of the PBD drug linker in DMSO isplaced in a suitable container for dilution with propylene glycol priorto addition to the reaction. To maintain solubility of the PBD druglinker, the antibody itself is first diluted with propylene glycol to afinal concentration of 33% (e.g., if the antibody solution was in a 60mL reaction volume, 30 mL of propylene glycol was added). This samevolume of propylene glycol (30 mL in this example) is added to the PBDdrug linker as a diluent. After mixing, the solution of PBD drug linkerin propylene glycol is added to the antibody solution to effect theconjugation; the final concentration of propylene glycol is 50%. Thereaction is allowed to proceed for 30 minutes and then quenched byaddition of 5 equivalents of N-acetyl cysteine. The ADC is purified byultrafiltration through a 30 kD membrane. (Note that the concentrationof propylene glycol used in the reaction can be reduced for anyparticular PBD, as its sole purpose is to maintain solubility of thedrug linker in the aqueous media.)

For halo-acetamide-based Linker-Drug compounds, conjugation can beperformed generally as follows. To a solution of reduced and reoxidizedantibodies (having introduced cysteines in the heavy chain) in 10 mMTris (pH 7.4), 50 mM NaCl, and 2 mM DTPA is added 0.5 volumes ofpropylene glycol. A 10 mM solution of acetamide-based Linker-Drugcompound in dimethylacetamide is prepared immediately prior toconjugation. An equivalent amount of propylene glycol as added to theantibody solution is added to a 6-fold molar excess of the Linker-Drugcompound. The dilute Linker-Drug solution is added to the antibodysolution and the pH is adjusted to 8-8.5 using 1 M Tris (pH 9). Theconjugation reaction is allowed to proceed for 45 minutes at 37° C. Theconjugation is verified by reducing and denaturing reversed phase PLRP-Schromatography. Excess Linker-Drug compound is removed with Quadrasil MPresin and the buffer is exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl,and 5% propylene glycol using a PD-10 desalting column.

Illustrative Synthesis Schemes for Drug Linkers

The following schemes are illustrative of routes for synthesising druglinkers—the PBD dimer is shown with specific substituents, and dimerlinks, but these may be varied within the scope of the presentinvention.

where Prot Sub refers to either the OH or CO₂H phenyl substituent groupsor their protected versions. The protection may be installed in light ofthe reactions carried out to introduce the linking unit, and may beremoved when appropriate during the synthesis. In some embodiments,protection would be in place for step (i), but would be removed eitherbefore or after step (ii). In other embodiments, protection would be inplace for step (i), but would be removed either after step (iii).

The glucuronide linker intermediate S1 (reference: Jeffrey et al.,Bioconjugate Chemistry, 2006, 17, 831-840) can be treated withdiphosgene in dichlroromethane at −78° C. to afford the glucuronidechloroformate, which is then reacted with the PBD dimer S2 dissolved inCH₂Cl₂ by dropwise addition. Warming the reaction to 0° C. over 2 hoursfollowed by extraction will yield the compound S3. Treating a solutionof S3 in an equal solvent mixture of MeOH, tetrahydrofuran, and water(cooled to 0° C.) with lithium hydroxide monohydrate for 4 hours,followed by reaction with glacial acetic acid will yield the compoundS4. Adding maleimidocaproyl NHS ester to a solution of S4 in DMF,followed by diisopropylethylamine and stirring at room temperature undernitrogen for 2 hours will yield the desired drug linker S5.

This approach could also be used with PBD dimers containing aliphaticamines, such as benzylamine, e.g. S6:

The methods of Examples 2 and 3 could also be applied to a wide varietyof the PBD dimers of the present invention in order to introducepeptidic linkers.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In some embodiments, R^(6′), R^(7′), R^(9′), R^(10′), and Y′ arepreferably the same as R⁶, R⁷, R⁹, R¹⁰, R¹¹ and Y respectively.

Dimer Link

Y and Y′ are preferably O.

R″ is preferably a C₃₋₇ alkylene group with no substituents. Morepreferably R″ is a C₃, C₅ or C₇ alkylene. Most preferably, R″ is a C₃ orC₅ alkylene.

R⁶ to R⁹

R⁹ is preferably H.

R⁶ is preferably selected from H, OH, OR, SH, NH₂, nitro and halo, andis more preferably H or halo, and most preferably is H.

R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, andhalo, and more preferably independently selected from H, OH and OR,where R is preferably selected from optionally substituted C₁₋₇ alkyl,C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R may be more preferably aC₁₋₄ alkyl group, which may or may not be substituted. A substituent ofinterest is a C₅₋₆ aryl group (e.g. phenyl). Particularly preferredsubstituents at the 7-positions are OMe and OCH₂Ph. Other substituentsof particular interest are dimethylamino (i.e. —NMe₂); —(OC₂H₄)_(q)OMe,where q is from 0 to 2; nitrogen-containing C₆ heterocyclyls, includingmorpholino, piperidinyl and N-methyl-piperazinyl.

These preferences apply to R^(9′), R^(6′) and R^(7′) respectively.

R²

A in R² may be phenyl group or a C₅₋₇ heteroaryl group, for examplefuranyl, thiophenyl and pyridyl. In some embodiments, A is preferablyphenyl. In other embodiments, A is preferably thiophenyl, for example,thiophen-2-yl and thiophen-3-yl.

X is a group selected from NHR^(N), wherein R^(N) is selected from thegroup comprising H and C₁₋₄ alkyl,

In some embodiments, X may preferably be NHR^(N). X may more preferablybe NHMe, NHEt, and NH₂, and may even more preferably be: NH₂.

Q²-X may be on any of the available ring atoms of the C₅₋₇ aryl group,but is preferably on a ring atom that is not adjacent the bond to theremainder of the compound, i.e. it is preferably β or γ to the bond tothe remainder of the compound. Therefore, where the C₅₋₇ aryl group (A)is phenyl, the substituent (Q²-X) is preferably in the meta- orpara-positions, and more preferably is in the para-position.

In some embodiments, Q¹ is a single bond. In these embodiments, Q² isselected from a single bond and —Z—(CH₂)_(n)—, where Z is selected froma single bond, O, S and NH and is from 1 to 3. In some of theseembodiments, Q² is a single bond. In other embodiments, Q² is—Z—(CH₂)_(n)—. In these embodiments, Z may be O or S and n may be 1 or nmay be 2. In other of these embodiments, Z may be a single bond and nmay be 1.

In other embodiments, Q¹ is —CH═CH—.

In some embodiments, R² may be -A-CH₂—X and -A-X. In these embodiments,X may preferably be NH₂.

R¹²

R¹² may be a C₅₋₇ aryl group. A C₅₋₇ aryl group may be a phenyl group ora C₅₋₇ heteroaryl group, for example furanyl, thiophenyl and pyridyl. Insome embodiments, R¹² is preferably phenyl. In other embodiments, R¹² ispreferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.

R¹² may be a C₈₋₁₀ aryl, for example a quinolinyl or isoquinolinylgroup. The quinolinyl or isoquinolinyl group may be bound to the PBDcore through any available ring position. For example, the quinolinylmay be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl,quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yland quinolin-6-yl may be preferred. The isoquinolinyl may beisoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl,isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of theseisoquinolin-3-yl and isoquinolin-6-yl may be preferred.

R¹² bears a substituent selected from OH, CO₂H, CO₂R^(O), where R^(O) isselected from C₁₋₄ alkyl. The substituent may be any position.

Where R¹² is C₅₋₇ aryl group, a single substituent is preferably on aring atom that is not adjacent the bond to the remainder of thecompound, i.e. it is preferably β or γ to the bond to the remainder ofthe compound. Therefore, where the C₅₋₇ aryl group is phenyl, thesubstituent is preferably in the meta- or para-positions, and morepreferably is in the para-position.

Where R¹² is a C₈₋₁₀ aryl group, for example quinolinyl orisoquinolinyl, it may bear any number of substituents at any position ofthe quinoline or isoquinoline rings.

R^(O) is preferably selected from C₁₋₂ alkyl, i.e. methyl and ethyl.

R¹² groups

Particularly preferred substituted R¹² groups include, but are notlimited to, 4-hydroxyphenyl, 3-hydroxyphenyl, 4-carboxy-phenyl,3-carboxy-phenyl, 4-methyloxycarbonyl-phenyl,3-methyloxycarbonyl-phenyl, 4-ethyloxycarbonyl-phenyl and4-ethyloxycarbonyl-phenyl.

M and z

It is preferred that M and M′ are monovalent pharmaceutically acceptablecations, and are more preferably Na⁺.

z is preferably 3.

Accordingly, compounds of the present invention include, for example,those of formula I, or a pharmaceutically acceptable salt or solvatethereof, wherein

(i) R² is of formula III:

where A is a phenyl group, X is NHR^(N), wherein R^(N) is selected fromthe group comprising H and C₁₋₄ saturated alkyl, Q¹ is a single bond,and the remainder of the substituents are as defined herein.

Compounds of the present invention include, for example, those offormula I, or a pharmaceutically acceptable salt or solvate thereof,wherein

(ii) R² is of formula III:

where A is a phenyl group, X is NHR^(N), wherein R^(N) is selected fromthe group comprising H and C₁₋₄ saturated alkyl, Q¹ is a single bond, Q²is selected from a single bond and —Z—(CH₂)_(n)—, where Z is selectedfrom a single bond and n is from 1 to 3; and the remainder of thesubstituents are as defined herein.(iii) Compounds of the present invention include, for example, those offormula I, or a pharmaceutically acceptable salt or solvate thereof,wherein R¹² is a phenyl group, substituted by a group selected fromCO₂H, CO₂R^(O), where R^(O) is selected from saturated C₁₋₄ alkyl; andthe remainder of the substituents are as defined herein.(iv) Compounds of the present invention include, for example, those offormula I, or a pharmaceutically acceptable salt or solvate thereof,wherein R¹² is a phenyl group, substituted by a group selected fromCO₂H, CO₂R^(O), where R^(O) is selected from methyl or ethyl; and theremainder of the substituents are as defined herein.(v) Compounds of the present invention include, for example, those offormula I, or a pharmaceutically acceptable salt or solvate thereof,whereinR² is of formula III:

where A is a phenyl group, X is NHR^(N), wherein R^(N) is selected fromthe group comprising H and C₁₋₄ saturated alkyl, Q¹ is a single bond, Q²is selected from a single bond and —Z—(CH₂)_(n)—, where Z is selectedfrom a single bond and n is from 1 to 3; R¹² is a phenyl group,substituted by a group selected from OH, CO₂H, CO₂R^(O), where R^(O) isselected from C₁₋₄ saturated alkyl; and the remainder of thesubstituents are as defined herein.(vi) Compounds of the present invention include, for example, those offormula I, or a pharmaceutically acceptable salt or solvate thereof,whereinR² is of formula III:

where A is a phenyl group, X is NHR^(N), wherein R^(N) is selected fromthe group comprising H and C₁₋₄ saturated alkyl, Q¹ is a single bond, Q²is selected from a single bond and —Z—(CH₂)_(n)—, where Z is selectedfrom a single bond and n is from 1 to 3; R¹² is a phenyl group,substituted by a group selected from CO₂H, CO₂R^(O), where R^(O) isselected from methyl or ethyl; and the remainder of the substituents areas defined herein.

Preferred compounds of the present invention include any of thosedescribed in (i) through (vi) wherein:

(a) the substituent group on R¹² is in the meta- or para-position, andmore preferably in the para-position,

(b) Y and Y′ are O,

(c) R″ is —(CH₂)—(CH₂)—(CH₂)— or —(CH₂)—(CH₂)—(CH₂)—(CH₂)—(CH₂)—,(d) R¹⁰ and R¹¹ form a nitrogen-carbon bond between the nitrogen andcarbon atoms to which they are bound and R^(10′) and R^(11′) form anitrogen-carbon bond between the nitrogen and carbon atoms to which theyare bound,(e) R⁷ is methoxy or ethoxy and R^(7′) is methoxy or ethoxy, or(f) R⁶, R⁹, R^(6′), and R^(9′) are hydrogen, or any combination of (a)through (f).

Particularly preferred compounds of the present invention are of formulaIa:

or a pharmaceutically acceptable salt or solvate thereof, wheren is 1 or 3;R^(1a) is methyl or phenyl;

R^(2a) is:

where R^(N) is selected from H and methyl;R^(12a) is selected from:

Particularly preferred compounds include:

or a pharmaceutically acceptable salt or solvate thereof.

3^(rd) Aspect

The preferences expressed above for the first aspect may apply to thecompounds of this aspect, where appropriate.

When R¹⁰ is carbamate nitrogen protecting group, it may preferably beTeoc, Fmoc and Troc, and may more preferably be Troc.

When R¹¹ is O-Prot^(O), wherein Prot^(O) is an oxygen protecting group,Prot^(O) may preferably be TBS or THP, and may more preferably be TBS.

When R¹⁰ is a hemi-aminal nitrogen protecting group, it may preferablybe MOM, BOM or SEM, and may more preferably be SEM.

The preferences for compounds of formula I apply as appropriate to D inthe sixth aspect of the invention. For example, in the sixth aspect, thePBD dimer is any of the compounds of formula I, or a pharmaceuticallyacceptable salt or solvate thereof, described herein expect that,

is replaced with

is replaced with

and *—NHR^(N) is replaced with

where the wavy line indicates the point of attachment to the LinkerUnit.

Accordingly, the Conjugates of the present invention include thosehaving the following formula (IV)

L-(LU-D)_(p)  (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), LU is a Linker unit and the PBDdimer D is any of the compounds of formula I, or a pharmaceuticallyacceptable salt or solvate thereof, described herein expect that,

is replaced with

is replaced with

and *—NHR^(N) is replaced with

where the wavy line indicates the point of attachment to the LinkerUnit.(a) Conjugates of the present invention include, for example, those ofthe formula:

CBA-A¹-L¹-*

-   -   where the asterisk indicates the point of attachment to the PBD        dimer (D) or the Spacer unit, CBA is the Cell Binding Agent, L¹        is a Specificity unit that is cleavable by the action of an        enzyme, and A¹ is a Stretcher unit connecting L¹ to the Cell        Binding Agent.        (b) Conjugates of the present invention include, for example,        those of the formula:

CBA-A¹-*

-   -   where the asterisk indicates the point of attachment to the PBD        dimer (D), CBA is the Cell Binding Agent, L¹ and A¹ is a        Stretcher unit connecting the Drug to the Cell Binding Agent.        (c) Conjugates of the present invention include, for example,        those of the formula:

CBA-A¹-L¹-*

-   -   where the asterisk indicates the point of attachment to the PBD        dimer (D), CBA is the Cell Binding Agent, A¹ is a Stretcher unit        connecting L¹ to the Cell Binding Agent and L¹ is a Specificity        unit that is cleavable by the action of cathepsin, L¹ is a        dipeptide, L¹ is a dipeptide that is cleavable by the action of        cathepsin or L¹ is a dipeptide selected from -Phe-Lys-,        -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.

Preferred conjugates of the present invention include any of thosedescribed in (a)-(c) wherein A¹ is

-   -   where the asterisk indicates the point of attachment to L¹ or D,        the wavy line indicates the point of attachment to CBA, and n is        0 to 6 (preferably n is 5).

Particularly preferred conjugates of the present invention are offormula Ib, Ic, 1d, and 1e:

or a pharmaceutically acceptable salt or solvate thereof, wheren is 1 or 3;R^(1a) is methyl;

R^(N) is H

R^(12a) is selected from:

A¹ is a Stretcher unit;L¹ is a dipeptide that is cleavable by the action of cathepsin;Ab is an antibody; andp is from 1 to 20.

In a particularly preferred embodiment of formulas Ib, Ic, Id, and Ie,or a pharmaceutically acceptable salt or solvate thereof, the connectionbetween the antibody and the Linker Unit is formed between a thiol groupof a cysteine residue of the antibody and a maleimide group of theLinker unit.

Particularly preferred conjugates include:

or a pharmaceutically acceptable salt or solvate thereof, whereinn is 1 or 3.A¹ is a Stretcher unit;L¹ is a dipeptide that is cleavable by the action of cathepsin;Ab is an antibody; andp is from 1 to 20.

In a particularly preferred embodiment, for all of these preferredconjugates, the connection between the antibody and the Linker is formedbetween a thiol group of a cysteine residue of the antibody and amaleimide group of the Linker unit.

In a particularly preferred embodiment, for all of these preferredconjugates, the antibody is a monoclonal antibody that specificallybinds to the Cripto antigen, CD19 antigen, CD20 antigen, CD22 antigen,CD30 antigen, CD33 antigen, Glycoprotein NMB, CanAg antigen, Her2(ErbB2/Neu) antigen, CD56 (NCAM) antigen, CD70 antigen, CD79 antigen,CD138 antigen, PSCA, PSMA (prostate specific membrane antigen), BCMA,E-selectin, EphB2, Melanotransferin, Muc16 antigen or TMEFF2 antigen.

The preferences for compounds of formula I, or a pharmaceuticallyacceptable salt or solvate thereof, apply as appropriate to D in theseventh aspect of the invention. For example, in the seventh aspect, thePBD dimer is any of the compounds of formula I, or a pharmaceuticallyacceptable salt or solvate thereof, described herein expect that, expectthat,

is replaced with

is replaced with

and *—NHR^(N) is replaced with

where the wavy line indicates the point of attachment to the LinkerUnit.

Particularly preferred Drug-Linkers of the present invention are offormula If, Ig, Ih and Ii:

or a pharmaceutically acceptable salt or solvate thereof, wheren is 1 or 3;R^(1a) is methyl or phenyl;

R^(N) is H

R^(12a) is selected from:

A¹ is a Stretcher unit; andL¹ is a dipeptide that is cleavable by the action of cathepsin.

Particularly preferred drug-linkers include:

or a pharmaceutically acceptable salt or solvate thereof, whereinn is 1 or 3.A¹ is a Stretcher unit; andL¹ is a dipeptide that is cleavable by the action of cathepsin.

EXAMPLES General Experimental Methods for Example 1

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40-60° C.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run usinga mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B)(formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then5% B to 95% B within 3 min. The composition was held for 0.5 min at 95%B, and then returned to 5% B in 0.3 minutes. Total gradient run timeequals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero deadvolume tee piece which passes into the mass spectrometer. Wavelengthdetection range: 220 to 400 nm. Function type: diode array (535 scans).Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm

Example 1

(a)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentoxyoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(2)

1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](1)(Compound 8b in WO 2010/043880) (2.8 g, 2.4 mmol, 1 eq) was added toa mixture of sodium carbonate (388 mg, 3.66 mmol, 1.52 eq) and4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)aniline (509 mg, 2.32mmol, 0.95 eq), in toluene/water/ethanol (20 mL/10 mL/10 mL). Thereaction flask was flushed with argon and solid Pd(0)tetrakistriphenylphosphine (84 mg, 0.072 mmol, 0.03 eq) was added. The reactionwas allowed to proceed for 2 hours at 26° C. with vigorous stirringunder argon. The mixture was partitioned between ethyl acetate (200 mL)and water (100 mL). The organic phase was washed with water (100 mL),followed by brine (50 mL). The organic phase was dried over magnesiumsulphate and the volatiles removed by rotoevaporation, followed by hardvacuum. The residue was purified by flash chromatography (gradient ethylacetate/hexane, 30/70 up 100/0, v/v). The unsymmetrical amino triflate(2) was isolated in 46% yield (1.23 g). LC/MS rt 3.80 min m/z (1087.6)M+H. 932 mg (33%) of starting material and 400 mg (16%) of symmetrical4-amino phenyl product were also obtained.

(b)(S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]diazepin-2-yltrifluoromethanesulfonate (3)

The amino triflate (2) was dissolved in dry THF (15 mL) and cooled at−78° C. (1.2 g, 1.1 mmol, 1 eq). A solution of super hydride in THF (1M, 3.3 mL, 3.3 mmol, 3 eq) was injected slowly in the stirred reactionmixture. Reaction completion was observed after 15 minutes. The reactionmixture was quenched with water (10 mL) and later extracted with DCM (50mL). The organics were washed with water (100 mL), then brine (50 mL).The organic phase was dried over magnesium sulphate and the volatilesremoved by rotoevaporation, followed by hard vacuum. The crudecarbinolamine (3) (1.10 g) was not purified and used directly in thenext step. LC/MS rt 2.68 min m/z (796) M+H for SEM deprotected imine(self-immolation under the acidic conditions of the LC/MS).

(c)(S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methyloxycarbonylphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one(4)

The crude SEM protected carbinolamine triflate (3) obtained in theprevious step (1.10 g, 1 mmol, 1 eq) was added to a mixture of sodiumcarbonate (341 mg, 3.2 mmol, 3.2 eq) and phenylboronic acid methyl ester(286 mg, 1.6 mmol, 1.6 eq), in toluene/water/methanol/THF (10 mL/5 mL/5mL/5 mL). The reaction flask was flushed with argon and solidPd(0)tetrakis triphenylphosphine (35 mg, 0.030 mmol, 0.03 eq) was added.The reaction was allowed to proceed overnight with vigorous stirringunder argon. The mixture was partitioned between ethyl acetate (200 mL)and water (100 mL). The organic phase was washed with water (100 mL),followed by brine (50 mL). The organic phase was dried over magnesiumsulphate and the volatiles removed by rotoevaporation, followed by hardvacuum. The residue was treated with DCM (50 mL), ethanol (140 mL),water (70 mL) and silica gel (100 g). The viscous mixture was allowed tostir at room temperature for 3 days. The mixture was filtered slowlythrough a sinter funnel and the silica residue washed with 90/10chloroform/methanol v/v (500 mL). The organic phase was washed withwater (300 mL), brine (100 mL), dried (magnesium sulphate), filtered,and evaporated in vacuo to provide the crude material which was purifiedby flash chromatography (gradient methanol/chloroform, 0/100 up 4/96,v/v) to yield 200 mg (25%) of PBD dimer LC/MS rt 2.68 min m/z (782) M+H.

General Experimental Methods for Examples 2 to 3

All commercially available anhydrous solvents were used without furtherpurification. Analytical thin layer chromatography was performed onsilica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, N.J.).Radial chromatography was performed on Chromatotron apparatus (HarrisResearch, Palo Alto, Calif.). Analytical HPLC was performed on a VarianProStar 210 solvent delivery system configured with a Varian ProStar 330PDA detector. Samples were eluted over a C12 Phenomenex Synergi 2.0×150mm, 4 μm, 80 Å reverse-phase column. The acidic mobile phase consistedof acetonitrile and water both containing 0.1% formic acid. Compoundswere eluted with a linear gradient of acidic acetonitrile from 5% at 1min post injection, to 95% at 11 min, followed by isocratic 95%acetonitrile to 15 min (flow rate=1.0 mL/min). LC-MS was performed on aZMD Micromass mass spectrometer interfaced to an HP Agilent 1100 HPLCinstrument equipped with a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Åreverse phase column. The acidic eluent consisted of a linear gradientof acetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min,followed by isocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min).Preparative HPLC was carried out on a Varian ProStar 210 solventdelivery system configured with a Varian ProStar 330 PDA detector.Products were purified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm,80 Å reverse phase column eluting with 0.1% formic acid in water(solvent A) and 0.1% formic acid in acetonitrile (solvent B). Thepurification method consisted of the following gradient of solvent A tosolvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min;followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min withmonitoring at 254 nm.

Example 2

(a)(S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoicacid (5)

Maleimidocaproyl N-hydroxysuccinimide (1.619 g, 5.25 mmol, 1.05 eq.) andH-Val-Ala-OH (0.941 g, 5 mmol, 1 eq.) were placed in a 25 mL recoveryflask with a stir bar and the flask was flushed with nitrogen. DMF (4.7mL) was added and the resulting white slurry was stirred. DIPEA (0.87mL, 5 mmol, 1 eq) was added and the mixture was allowed to stir at roomtemperature overnight. The mixture was cooled in an ice/water bath and2M HCl (3 mL, 6 mmol) was added dropwise. The viscous mixture wastransferred to a separatory funnel and the reaction vessel rinsed withsat. NaCl (7 mL), EtOAc (10 mL), sat NaCl (10 mL) and EtOAc (5 mL).After separation of the aqueous phase, it was extracted with additionalEtOAc (2×15 mL). The combined organic extracts were washed with sat NaCl(4×15 mL), until the washings were pH ˜3.5. The organic extracts weredried over Na2SO4, filtered and concentrated under reduced pressure togive crude 5 as a white solid (2.172 g, 114% crude yield). Crude 5 wassuspended in warm CH₂Cl₂ (35 mL) and filtered to remove a fine whitesolid. The solids were rinsed with additional CH₂Cl₂ (3 mL). Toluene (5mL) was added and the mixture was cooled in an ice/water bath, whichresulted in a thick slurry. The solids were collected by filtration,washed with a cold mixture of CH₂Cl₂ (12 mL) and toluene (2 mL) anddried by pulling air through the sample overnight to give 5 as a whitesolid (1.327 g, 70% yield). TLC: Rf=0.26, 10% MeOH in CH₂Cl₂. 1H NMR(CDCl₃) (ppm) 0.95 (d, J=17 Hz, 3H), 0.98 (d, J=17 Hz, 3H), 1.30 (m,2H), 1.40 (d, J=17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1H), 2.25 (dt, J=4,19 Hz, 2H), 3.35 (s, 1H), 3.49 (t, J=17 Hz, 2H), 4.20 (d, J=18 Hz, 1H),4.38 (m, 1H), 6.80 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.05min. LC-MS: t_(R) 11.17 min, m/z (ES+) found 381.9 (M+H)+, m/z (ES−)found 379.9 (M−H)−.

(b) Methyl4-((S)-8-((5-(((S)-2-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoate(6)

A 10 mL flask was charged with 5 (11 mg, 29 μmol), EEDQ (8.9 mg, 36μmol), and 0.46 mL anhydrous CH₂Cl₂. Methanol (24 μL) was added tofacilitate dissolution and the mixture was stirred under nitrogen for 15min. Aniline 4 (18 mg, 24 μmol) was then added and the reaction mixturewas stirred at room temperature for 4 hours, at which time LC-MSrevealed conversion to product. The reaction was concentrated, dissolvedin CH₂Cl₂ (1 mL) and purified by radial chromatography on a 1 mmchromatotron plate eluted with CH₂Cl₂/CH₃OH mixtures (100:0 to 90:10CH₂Cl₂/CH₃OH) to provide 6 (9.9 mg, 36%). Analytical HPLC: t_(R) 12.10min. LC-MS: t_(R) 12.91 min, m/z (ES⁺) found 1145.6 (M+H)⁺.

Example 3

9: R¹═H, R²═CH₃10: R¹═H, R²═H11: R¹=MC, R²═H

Compound 7 was prepared in a similar fashion to compound 5 in Example2(a) using allyl chloroformate in place of maleimidocaproylN-hydroxysuccinimide and dichloromethane as the reaction solvent.

(a) Methyl4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoate(8)

To a 7 (52 mg, 0.192 mmol) in 5% methanol/dichloromethane (3 mL) was at0° C. was added EEDQ (47 mg, 0.193 mmol) and the mixture was stirred for15 minutes before addition of 4 (50 mg, 0.064 mmol). The reactionmixture was allowed to warm to an ambient temperature and was monitoredby LC-MS. The mixture was aspirated onto a 1 mm radial chromatotronplate and eluted with 1 to 3% methanol/dichloromethane. Productcontaining fractions were combined and concentrated to give 43 mg (65%)of 8 as a yellow solid: MS (ES⁺) m/z 1036.87 [M+H]⁺.

(b) Methyl4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoate(9)

To a solution of 8 (43 mg) in anhydrous dichloromethane (3 mL) was addedPh₃P (0.5 mg, 0.002 mmol), pyrollidine (7 μL, 0.082 mmol) and tetrakispalladium (1.1 mg, 0.001 mmol). After approximately 30 minutes, thereaction mixture was aspirated onto a 1 mm radial chromatotron plate andeluted with 5% and then 10% methanol in dichloromethane. The major bandwas collected and concentrated under reduced pressure to give 22 mg(56%) of 9: MS (ES⁺) m/z 952.5 [M+H]⁺.

(c)4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoicacid (10)

To 9 (20 mg) in THF/CH₃OH (2 mL) was added a lithium hydroxide solution(1 mL of a 0.1 M solution). The reaction mixture was stirred at anambient temperature. At 5 hours, LC-MS revealed approximately a 30%conversion to desired product with significant decomposition. Thereaction mixture was cooled to −80° C. for 16 hours. LC-MS showed a ˜1:1mixture of 10 and 9. The reaction mixture was neutralized with 0.1N HCl(˜1 mL) and was concentrated to approximately 1 mL. DMSO (1 mL) andCH₃CN (1 mL) were added, and the mixture was purified by preparatoryreverse-phase HPLC. Product containing fractions were combined, frozenand lyophilized. This resulted in 1.7 mg (9%) of 10 as a yellow film: MS(ES⁺) m/z 938 [M+H]⁺.

(d)4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoicacid (11)

To a mixture of 10 (1.7 mg, 1.8 μmol) in DMF (100 μL) was added DIPEA (1μL, 5.75 μmol) and maleimidocaproyl-NHS ester (4.6 mg, 15 □mol). Thereaction was monitored by LC-MS. After 1 hour, the reaction mixture wasconcentrated under reduced pressure, dissolved in 0.5 mL of DMSO, 0.5 mLof acetonitrile and 0.5 mL of water, and purified by preparativereverse-phase HPLC. The product containing fraction was frozen andlyophilized to give 0.2 mg (10%) of 11: MS (ES⁺) m/z 1131.6 [M+H]⁺.

Example 4

(a)(S)-2-(4-aminophenyl)-8-(3-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione(13)

A flask was charged with aniline triflate 12 (compound 9, WO 2011/130613A1) (520 mg, 490 μmol, 1 eq) dissolved in toluene (5.4 mL), ethanol (2.7mL), and water (2.7 mL). To the stirred solution was added4-hydroxyphenylboronic acid (88 mg, 640 μmol, 1.3 eq), sodium carbonate(83 mg, 780 μmol, 1.6 eq), and tetrakis(triphenylphosphine)palladium(0)(23 mg, 20 μmol, 0.04 eq), the reaction was stirred vigorously overnightat room temperature under nitrogen. After 22 hours the reaction hadstalled. Additional tetrakis(triphenylphosphine)palladium(0) (100 mg, 87μmol, 0.18 eq) and 4-hydroxyphenylboronic acid (88 mg, 640 μmol, 1.3 eq)were added and the reaction was stirred at 35° C. for an additional 24hours, at which time LC/MS revealed conversion to product. The reactionwas concentrated and then partitioned between ethyl acetate (100 mL) andwater (100 mL). The aqueous layer was extracted two times with ethylacetate (100 mL). The organic layer was then washed with water (100 mL),brine (100 mL), dried over sodium sulfate, and concentrated to drynessto provide crude SEM dilactam 13. The crude product was purified byflash chromatography, eluting with mixtures of hexanes:ethyl acetate(75:25 to 0:100), to provide pure product 13 (218 mg, 44%). LC-MS: t_(R)11.54 min, m/z (ES⁺) found 1004.3 (M+H)⁺. ¹H NMR (CDCl₃) δ (ppm) 0.02(s, 18H), 0.98 (m, 4H), 2.44 (m, 2H), 3.12 (m, 2H), 3.67 (m, 3H), 3.77(m, 4H), 3.91 (m, 8H), 4.29 (t, J=5.9 Hz, 4H), 4.59 (dt, J=3.1, 10.2 Hz,2H), 4.76 (dd, J=3.1, 10.2 Hz, 2H), 5.52 (d, J=10.2 Hz, 2H), 6.34 (bs,1H), 6.66 (d, J=8.2 Hz, 2H), 6.83 (d, J=8.6 Hz, 2H), 7.22 (m, 4H), 7.27(m, 6H), 7.39 (s, 2H).

(b)(S)-2-(4-aminophenyl)-8-(3-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one(14)

A flame-dried flask was charged with SEM dilactam 13 (109 mg, 109 μmol,1 eq) dissolved in anhydrous tetrahydrofuran (2.2 mL), and cooled to−78° C. Lithium triethylborohydride (0.33 mL of a 1 M solution in THF,330 μmol, 3 eq) was added dropwise and the reaction was stirred undernitrogen for 2.5 hours, at which time LC revealed incomplete conversionto product. An additional 0.66 mL of reductant was added and thereaction was stirred for one more hour. The reaction was quenchedthrough the addition of water (1 mL) and allowed to warm to roomtemperature, then diluted brine (25 mL) and extracted three times withdichloromethane (25 mL). The combined organics were washed with brine(25 mL), dried over sodium sulfate, and evaporated to dryness. Theresidue was dissolved in a mixture of dichloromethane (2.8 mL), ethanol(7.4 mL), and water (1.0 mL), and silica gel (2.7 g) was added. Theresulting slurry was stirred at room temperature for 4 days. TLCanalysis revealed conversion to imine dimer 14, at which time the slurrywas filtered over a sintered glass funnel and the silica gel cake waswashed with 10% methanol in chloroform until no further PBD absorbancewas observed in the filtrate. Concentration of the filtrate providedcrude imine dimer 14. The material was dissolved in minimaldichloromethane and purified by radial chromatography on a 1 mmchromatotron plate eluted with CH₂Cl₂/MeOH mixtures (100:0 to 80:20) toprovide 14 (31 mg, 40%). LC-MS: t_(R) 8.48 min, m/z (ES⁺) found 712.2(M+H)⁺.

Example 5

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(15)

A flame-dried flask was charged with maleimidocaproyl-valine-alaninelinker (Compound 36 of Example 13 in WO 2011/130613 A1) (11 mg, 29 μmol,1.5 eq) dissolved in 0.8 mL of 5% methanol in anhydrous dichloromethane.The acid was pre-activated by addition ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (9 mg, 34 μmol, 1.8 eq),followed by stirring at room temperature under nitrogen for 15 minutes.The activated acid was then added to a flame-dried flask containing PBDdimer 14 (13 mg, 19 μmol, 1 eq). The reaction was stirred for 4 hours atroom temperature under nitrogen, at which time LC-MS revealed conversionto product. The material was diluted in dichloromethane and purified byradial chromatography on a 1 mm chromatotron plate eluted withCH₂Cl₂/MeOH mixtures (100:0 to 80:20) to provide 15 (7.7 mg, 38%).LC-MS: m/z (ES⁺) found 1075.5 (M+H)⁺.

Example 6 Preparation of PBD Dimer Conjugates

Antibodies with introduced cysteines: Antibodies to CD70 containing acysteine residue at position 239 of the heavy chain were fully reducedby adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to7.4 with 1M Tris buffer (pH 9.0). Following a 1 hour incubation at 37°C., the reaction was cooled to 22° C. and 30 equivalents ofdehydroascorbic acid were added to selectively reoxidize the nativedisulfides, while leaving cysteine 239 in the reduced state. The pH wasadjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction wasallowed to proceed for 1 hour at 22° C. The pH of the solution was thenraised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5equivalents of the PBD drug linker in DMSO were placed in a suitablecontainer for dilution with propylene glycol prior to addition to thereaction. To maintain solubility of the PBD drug linker, the antibodyitself was first diluted with propylene glycol to a final concentrationof 33% (e.g., if the antibody solution was in a 60 mL reaction volume,30 mL of propylene glycol was added). This same volume of propyleneglycol (30 mL in this example) was then added to the PBD drug linker asa diluent. After mixing, the solution of PBD drug linker in propyleneglycol was added to the antibody solution to effect the conjugation; thefinal concentration of propylene glycol is 50%. The reaction was allowedto proceed for 30 minutes and then quenched by addition of 5 equivalentsof N-acetyl cysteine. The ADC was then purified by ultrafiltrationthrough a 30 kD membrane. (Note that the concentration of propyleneglycol used in the reaction can be reduced for any particular PBD, asits sole purpose is to maintain solubility of the drug linker in theaqueous media.)

Example 7 Determination of Free Drug In Vitro Cytotoxicity

Cells as detailed below were collected and plated in 96 well black-sidedplates at a density of 10,000 cells/well in 150 μL of medium. Serialdilutions of the test article (50 μL) were added, and incubation wascarried out for 92 hours at 37° C. After addition of test compound,cultures were incubated to 96 hours at 37° C. Resazurin (0.25 mM, 50 μL,Sigma, St. Louis, Mo.) in medium was added and incubation was continuedfor 4 hours. The plates were read on a Fusion HT microplate reader(Packard, Meriden, Conn.) using an excitation wavelength of 525 nm andan emission wavelength of 590 nm. Data from all assays were reducedusing GraphPad Prism Version 4 for Windows (GraphPad Software, SanDiego, Calif.). The IC₅₀ concentrations compared to untreated controlcells were determined using a 4 parameter curve fits.

The IC₅₀ (pM) values for compounds 4 and 14:

TABLE 1 IC50 in pM following 48 hours treatment compound 786-O Caki-1HL60 HEL9217 4 50 20 8 8 14 200 400 30 50

Example 8 Determination of In Vitro Activity of Selected Conjugates

The in vitro cytotoxic activity of the selected antibody drug conjugateswas assessed using a resazurin (Sigma, St. Louis, Mo., USA) reductionassay (reference: Doronina et al., Nature Biotechnology, 2003, 21,778-784). The antibody drug conjugates were prepared as described abovein Example 6.

For the 96-hour assay, cells cultured in log-phase growth were seededfor 24 hours in 96-well plates containing 150 μL RPMI 1640 supplementedwith 20% FBS. Serial dilutions of ADC in cell culture media wereprepared at 4× working concentration; 50 μL of each dilution was addedto the 96-well plates. Following addition of ADC, the cells wereincubated with test articles for 4 days at 37° C. Resazurin was thenadded to each well to achieve a 50 μM final concentration, and theplates were incubated for an additional 4 hours at 37° C. The plateswere then read for the extent of dye reduction on a Fusion HT platereader (Packard Instruments, Meridien, Conn., USA) with excitation andemission wavelengths of 530 and 590 nm, respectively. The IC₅₀ value,determined in triplicate, is defined here as the concentration thatresults in a 50% reduction in cell growth relative to untreatedcontrols.

Referring to the tables below, the in vitro cytotoxicity of ADCs usingthe 96 hour assay is shown. The ADCs were tested against antigenpositive and antigen negative cell lines.

TABLE 2 IC50 in pM following 96 hours treatment antigen-negative ADCdrugs/Ab 786-O Caki-1 cell line h1F6ec-6 1.8 30 0.1 90,000 h1F6ec-11 1.850 30 No effect h1F6ec-15 2.0 30 13 10,000

Example 9 Determination of In Vivo Cytotoxicity of Selected Conjugates

All studies were conducted in accordance with the Animal Care and UseCommittee in a facility that is fully accredited by the Association forAssessment and Accreditation of

Laboratory Animal Care. ADC tolerability was first assessed to ensurethat the conjugates were tolerated at the doses selected for thexenograft experiments. BALB/c mice were treated with escalating doses ofADC formulated in PBS with 0.5 M arginine and 0.01% Tween 20. Mice weremonitored for weight loss and outward signs of morbidity followingtreatment; those that experienced greater than 20% weight loss ordisplayed signs of morbidity were euthanized. The antibody used was aCD70 antibody, humanized h1F6 (WO2006/113909), with a point mutationsubstituting cysteine for serine at position 239. Conjugation to theDrug Unit is through the introduced cysteine at position 239. An averageof 2 drugs is loaded per antibody.

In vivo therapy experiments were conducted in xenograft models in micebearing CD70+ renal cell carcinoma or non-Hodgkin lymphoma. Tumorfragments were implanted into nude mice. Mice were then randomized tostudy groups with each group averaging around 100 mm³. The ADCs wereadministered according to the schedule indicated. Tumor volume as afunction of time was determined using the formula (L×W²)/2. Animals wereeuthanized when tumor volumes reached 1000 mm³. Mice showing durableregressions were terminated around day 100 post implant.

FIG. 1 shows the results of treatment studies using h1F6ec-compound 6 inCD70+ renal cell carcinoma (786-O), with single dose given IP. In thefigure,

is untreated, • is treatment with h1F6ec-6 at 0.03 mg/kg and ∘ istreatment with h1F6ec-6 at 0.1 mg/kg.

FIG. 2 show the results of treatment studies using h1F6ec-compound 6 innon-Hodgkin lymphoma (MHHPreB1), with dosing q7dx2. In the figure,

is untreated and • is treatment with h1F6ec-6 at 0.1 mg/kg.

The results of a mouse tolerability experiment with h1F6ec-6 nominallyloaded at 2 drugs/mAb demonstrated that a single dose of 1 mg/kg waswell tolerated with no weight loss or signs of outward morbidity out to30 days. Administration of a higher dose (2.5 mg/kg) resulted in weightloss.

The IC₅₀ (nM) values for ADCs with Compound 6:

786-O Caki-1 CD70 neg CD70 neg CD70 neg cancer cancer cancer cancercancer ADCs cell line cell line cell line cell line cell line h1F6ec-6 10.5 7491 2074 5327 (1.8 dr/Ab)

The IC₅₀ (nM) values for ADCs with Compound 6 and Compound 11:

786-O Caki-1 CD70 neg CD70 neg CD70 neg cancer cancer cancer cancercancer ADCs cell line cell line cell line cell line cell line h1F6ec-114 2 No Effect 7725 Max (1.8 dr/Ab) Inh = 50% h1F6ec-6 2 0.01 7215 1415Max (1.8 dr/Ab) Inh = 45%

1. A Conjugate having formula IV:L-(LU-D)_(p)  (IV) or a pharmaceutically acceptable salt thereof;wherein L is a Ligand Unit selected from the group consisting of anantibody and an antigen binding fragment of an antibody; LU is a LinkerUnit of formula X:

wherein E is a glucuronic acid residue, the asterisk adjacent to thecarbonyl carbon atom indicates the point of attachment of that carbonatom to a Drug Unit, and the wavy line indicates the point of attachmentto the Ligand Unit, and -A¹- is selected from the group consisting of:

wherein n is 0 to 6;

wherein n is 0 or 1, and m is 0 to 30; and

wherein the asterisk indicates the point of attachment to the nitrogenatom of Formula X, and the wavy line indicates the point of attachmentto the Ligand unit; p is 1 to 20; and D is a Drug Unit, wherein the DrugUnit is a PBD compound of formula I:

wherein: A is C₅₋₇ aryl, wherein X is selected from the group consistingof:

wherein R^(N) is selected from the group consisting of H and C₁₋₄ alkyl,the asterisk indicates the point of attachment to Q², and the wavy lineindicates the point of attachment to the Linker Unit; and either (i) Q¹is a single bond, and Q² is selected from the group consisting of asingle bond and —Z—(CH₂)_(n)—, wherein Z is selected from the groupconsisting of a single bond, O, S and NH, and subscript n is from 1 to3, or (ii) Q¹ is —CH═CH—, and Q² is a single bond; and R¹² is C₅₋₁₀ arylhaving a substituent selected from the group consisting of OH, CO₂H, andCO₂R^(O), wherein R^(O) is C₁₋₄ alkyl; R⁶ and R⁹ are independentlyselected from the group consisting of H, R, OH, OR, SH, SR, NH₂, NHR,NRR′, nitro, Me₃Sn and halo; R⁷ is selected from the group consisting ofH, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo, wherein Rand R′ are independently selected from the group consisting ofoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl;and either: (a) R¹⁰ is H, and R¹¹ is OH or OR^(A), wherein R^(A) is C₁₋₄alkyl, or (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between thenitrogen and carbon atoms to which they are bound, or (c) R¹⁰ is H andR¹¹ is SO_(z)M, wherein subscript z is 2 or 3 and M is a monovalentpharmaceutically acceptable cation; and R″ is C₃₋₁₂ alkylene whosecarbon chain is optionally interrupted by one or more heteroatomsselected from the group consisting of O, S, and NR^(N2), wherein R^(N2)is H or C₁₋₄ alkyl, and/or by an aromatic ring; Y and Y′ are selectedfrom the group consisting of O, S, and NH; R^(6′), R^(7′), R^(9′) areselected from the same groups as R⁶, R⁷ and R⁹, respectively, andR^(10′) and R^(11′) are the same as R¹⁰ and R¹¹, respectively, whereinif R¹¹ and R^(11′) are SO_(z)M, then each M is a monovalentpharmaceutically acceptable cation or together is a divalentpharmaceutically acceptable cation; and wherein LU is attached to D viathe X substituent of R².
 2. The Conjugate of claim 1, wherein A¹ is:

wherein the asterisk adjacent to the carbonyl carbon atom indicates thepoint of attachment of that carbon atom to the nitrogen atom of FormulaX, and the wavy line indicates the point of attachment to the LigandUnit.
 3. The conjugate of claim 1 wherein the Drug Unit is selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof, wherein the asteriskadjacent to the nitrogen atom indicates the point of attachment of thatatom to LU.
 4. The conjugate of claim 1 wherein LU-D is

wherein Prot Sub is —OH or CO₂H, optionally protected, and the wavy lineindicates the point of attachment to L.
 5. The conjugate of claim 1wherein LU-D is

wherein Prot Sub is —OH or CO₂H, optionally protected, and the wavy lineindicates the point of attachment to L.
 6. A compound having formula:G¹-L¹-L²-D, or a pharmaceutically acceptable salt or solvate thereof,wherein -L¹-L²- has the structure of formula X′:

wherein E is a glucuronic acid residue, the asterisk indicates the pointof attachment to a Drug Unit, and the way line indicates the point ofattachment to G¹, wherein: G¹- is selected from the group consisting of:

wherein n is 0 to 6;

wherein n is 0 or 1, and m is 0 to 30; and

wherein the asterisk indicates the point of attachment to the nitrogenatom of Formula X, and the wavy line indicates the point of attachmentto the Ligand unit; and D is a Drug Unit, wherein the Drug Unit is a PBDcompound of formula I:

wherein: A is C₅₋₇ aryl, wherein X is selected from the group consistingof:

wherein R^(N) is selected from the group consisting of H and C₁₋₄ alkyl,the asterisk indicates the point of attachment to Q², and the wavy lineindicates the point of attachment to the Linker Unit; and either (i) Q¹is a single bond, and Q² is selected from the group consisting of asingle bond and —Z—(CH₂)_(n)—, wherein Z is selected from the groupconsisting of a single bond, O, S and NH, and subscript n is from 1 to3, or (ii) Q¹ is —CH═CH—, and Q² is a single bond; and R¹² is C₅₋₁₀ arylhaving a substituent selected from the group consisting of OH, CO₂H, andCO₂R^(O), wherein R^(O) is a C₁₋₄ alkyl; R⁶ and R⁹ are independentlyselected from the group consisting of H, R, OH, OR, SH, SR, NH₂, NHR,NRR′, nitro, Me₃Sn and halo; R⁷ is selected from the group consisting ofH, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo, wherein Rand R′ are independently selected from the group consisting ofoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl;and either: (a) R¹⁰ is H, and R¹¹ is OH or OR^(A), wherein R^(A) is C₁₋₄alkyl, or (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between thenitrogen and carbon atoms to which they are bound, or (c) R¹⁰ is H andR¹¹ is SO_(z)M, wherein subscript z is 2 or 3 and M is a monovalentpharmaceutically acceptable cation; and R″ is a C₃₋₁₂ alkylene whosecarbon chain is optionally interrupted by one or more heteroatomsselected from the group consisting of O, S, and NR^(N2), wherein R^(N2)is H or C₁₋₄ alkyl, and/or by an aromatic ring; Y and Y′ are selectedfrom the group consisting of O, S, and NH; R^(6′), R^(7′), R^(9′) areselected from the same groups as R⁶, R⁷ and R⁹, respectively, andR^(10′) and R^(11′) are the same as R¹⁰ and R¹¹, respectively, whereinif R¹¹ and R^(11′) are SO_(z)M, then each M is a monovalentpharmaceutically acceptable cation or together is a divalentpharmaceutically acceptable cation; and wherein LU is attached to D viathe X substituent of R².
 7. The compound of claim 6, wherein G¹ is:

wherein the asterisk adjacent to the carbonyl carbon atom indicates thepoint of attachment of that carbon atom to the nitrogen atom of FormulaX.
 8. The compound of claim 6 wherein the Drug Unit is selected from thegroup consisting of;

and pharmaceutically acceptable salts thereof, wherein the asteriskadjacent to the nitrogen atom indicates the point of attachment of thatatom to LU.
 9. The compound of claim 6 wherein LU-D is

wherein Prot Sub is —OH or CO₂H, optionally protected.
 10. The compoundof claim 6 wherein LU-D is

wherein Prot Sub is —OH or CO₂H, optionally protected, and the wavy lineindicates the point of attachment to L.
 11. A method treating aproliferative disease comprising administering to a subject in need oftreatment a therapeutically-effective amount of a conjugate of claim 1.